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Insights Into Peroxisome Function from the Structure of PEX3 In THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 33, pp. 25410–25417, August 13, 2010 © 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Insights into Peroxisome Function from the Structure of PEX3 in Complex with a Soluble Fragment of PEX19*□S Received for publication, April 27, 2010, and in revised form, May 17, 2010 Published, JBC Papers in Press, June 16, 2010, DOI 10.1074/jbc.M110.138503 Friederike Schmidt‡1, Nora Treiber§1,2, Georg Zocher‡, Sasa Bjelic¶, Michel O. Steinmetz¶, Hubert Kalbacher‡, Thilo Stehle‡ʈ3, and Gabriele Dodt‡4 From the ‡Interfaculty Institute for Biochemistry, University of Tu¨bingen, 72076 Tu¨bingen, Germany, the §Institute for Organic Chemistry and Biochemistry, University of Freiburg, 79106 Freiburg, Germany, the ¶Laboratory of Biomolecular Research, Structural Biology, Paul Scherrer Institut, 5232 Villigen PSI, Switzerland, and the ʈDepartment of Pediatrics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 The human peroxins PEX3 and PEX19 play a central role maintenance (6). Fifteen such proteins, which are named per- in peroxisomal membrane biogenesis. The membrane-an- oxins, are currently known in humans and the corresponding chored PEX3 serves as the receptor for cytosolic PEX19, genes (PEX genes) are highly conserved throughout the eukary- which in turn recognizes newly synthesized peroxisomal otic kingdom (7, 8). membrane proteins. After delivering these proteins to the All matrix proteins and most membrane proteins are Downloaded from peroxisomal membrane, PEX19 is recycled to the cytosol. The imported post-translationally into peroxisomes. The machin- molecular mechanisms underlying these processes are not ery of peroxins that mediates the import of matrix proteins well understood. Here, we report the crystal structure of the bearing a peroxisomal targeting signal is far better understood cytosolic domain of PEX3 in complex with a PEX19-derived than the machinery that mediates the recognition and import of peptide. PEX3 adopts a novel fold that is best described as a membrane proteins (9, 10). The peroxins PEX3,5 PEX16, and http://www.jbc.org/ large helical bundle. A hydrophobic groove at the membrane- PEX19 are known to be essential for peroxisomal membrane distal end of PEX3 engages the PEX19 peptide with nanomo- biogenesis as a loss of any of these proteins leads to the com- lar affinity. Mutagenesis experiments identify phenylalanine plete absence of detectable peroxisomal membrane structures 29 in PEX19 as critical for this interaction. Because key PEX3 (11). However, de novo formation of peroxisomes was observed residues involved in complex formation are highly conserved in cells deficient for each of these peroxins upon complemen- across species, the observed binding mechanism is of general tation with the wild type gene, raising an intriguing question at LIB4RI on December 18, 2018 biological relevance. about the origin of the peroxisomal membrane (11–15). The endoplasmic reticulum membrane as the obvious source was disputed for a long time as several studies indicate that this Peroxisomes are single membrane-bound organelles that process does not involve the classical coat protein I- and coat carry out a variety of metabolic processes. In addition to the protein II-dependent pathways (16–18). Recently, new evi- ␤ degradation of H2O2, the -oxidation of very long chain or dence for an involvement of the endoplasmic reticulum as a branched chain fatty acids and the synthesis of ether lipids are peroxisomal precursor has been reported in yeast (19–21) and performed in these subcellular compartments (1, 2). The bio- in mammalian cells (22–24), although the details of this process genesis of peroxisomes, including their formation and prolifer- remain to be elucidated. ation, as well as the degradation of peroxisomes are highly PEX19 is a farnesylated but hydrophilic protein that is pre- dynamic processes that are adapted to metabolic needs (3). dominantly found in the cytosol, with a smaller fraction tran- Defects in peroxisome biogenesis cause a number of severe siently located at the peroxisomal membrane (12, 25). In the inherited diseases, which are collectively referred to as peroxi- cytoplasm, PEX19 can act as a chaperone for newly synthesized some biogenesis disorders (4, 5). Studies in yeast and analysis of peroxisomal membrane proteins (PMPs) by binding them dur- patients affected by these disorders have led to the identifica- ing or after translation and keeping them in an import-compe- tion of specific proteins involved in peroxisomal formation and tent form (26, 27). For the majority of PMPs, the PEX19-bind- ing site matches the proposed membrane-targeting signal (11, * This work was supported by Deutsche Forschungsgemeinschaft Grants 28). Cargo-loaded PEX19 is directed to the peroxisomal mem- Do492/2 (to G. D.) and SFB685 (to H. K.). brane by docking to PEX3 (29). The predicted PEX3-binding The atomic coordinates and structure factors (code 3MK4) have been deposited domain of PEX19 is located within its first 56 amino acid resi- in the Protein Data Bank, Research Collaboratory for Structural Bioinformat- ics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/). dues, whereas the C-terminal part harbors the binding sites for □S The on-line version of this article (available at http://www.jbc.org) contains other peroxisomal membrane proteins (11, 26, 30, 31). After supplemental Figs. S1–S4 and Table S1. insertion of the PMP, PEX19 is released into the cytosol to 1 Both authors contributed equally to this work. initiate another import cycle (32). 2 Present address: Dept. of Cellular and Molecular Immunology, Max-Planck Institute of Immunobiology, 79108 Freiburg, Germany. 3 To whom correspondence may be addressed. Tel.: 49-7071-2973043; Fax: 5 The abbreviations used are: PEX, peroxisomal biogenesis factor; PMP, 49-7071-295565; E-mail: [email protected]. peroxisomal membrane protein; ITC, isothermal titration calorimetry; 4 To whom correspondence may be addressed. Tel.: 49-7071-2973349; Fax: HPLC, high pressure liquid chromatography; Bis-Tris, 2-(bis(2-hydroxy- 49-7071-295191; E-mail: [email protected]. ethyl)amino)-2-(hydroxymethyl)propane-1,3-diol. 25410 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285•NUMBER 33•AUGUST 13, 2010 Structure of an sPEX3-PEX19Pep Complex The peroxin PEX3 is anchored in the peroxisomal mem- 4 °C with 1 mg of tobacco etch virus protease/40 mg of protein brane via a short hydrophobic transmembrane segment while dialyzing against 1 liter of buffer A. A second Ni2ϩ col- within its N-terminal 33 residues, a region that is necessary umn removed residual uncleaved protein. Cleaved protein was and sufficient for targeting PEX3 to peroxisomes (33, 34). The then concentrated and applied to a SuperdexTM 200 10/300 cytosolic domain mediates the interaction with PEX19 (11). (GE Healthcare) size exclusion column using buffer C (50 mM PEX3 is imported into peroxisomes in a PEX19-independent Tris, 200 mM NaCl, 0.5 mM Tris-(2-carboxyethyl)phosphine, manner and hence defines a separate import pathway (26). The pH 8.0). Purity and homogeneity of the proteins were con- role of PEX16 during the import of peroxisomal membrane firmed by SDS-PAGE, native PAGE, and dynamic light scatter- proteins is less well defined, but it is thought to function as a ing. Protein folding was analyzed with circular dichroism spec- docking site for PEX3 (35). troscopy using a JASCO J-720 spectrophotometer. Protein To define the parameters that underlie the interaction of concentrations were determined by measurements of absorp- PEX3 with PEX19, we solved the structure of a soluble domain tion at 280 nm with a NanoDrop ND-1000 (PeqLab). of PEX3 in complex with a peptide corresponding to an N-ter- Peptide Synthesis—PEX19-derived peptides were prepared us- minal region of PEX19 (PEX19Pep). The soluble PEX3 domain ing solid-phase synthesis based on the N-(9-fluorenyl)methoxy- comprises residues 41–373 and contains a cysteine to serine carbonyl (Fmoc) strategy on a SyroII synthesizer (MultiSynTech, mutation at position 235 (sPEX3). In combination with affinity Witten, Germany) as described (36). Peptides were purified by measurements and mutagenesis experiments, this structure HPLC using a C18 column, resulting in a purity of 95%. Purity provides insights into the determinants of recognition of the and identity of the products were confirmed by analytical PEX3-PEX19 complex. As residues in the contact area are HPLC, matrix-assisted laser desorption/ionization time of Downloaded from highly conserved among eukaryotes, our structure can serve as flight mass spectrometry, and electrospray ionization mass a general model for understanding the functions of PEX3 and spectrometry. PEX19 in peroxisomal biogenesis. Moreover, the structure pre- Affinity Measurements Using Isothermal Titration Calorim- sented here provides one of the first views of any interaction etry (ITC)—Affinity measurements for PEX326–373(C235S) between two peroxins at high resolution. with full-length PEX19 were carried out in buffer C at 25 °C http://www.jbc.org/ with a VP-ITC system (Microcal). Purified PEX326–373(C235S) EXPERIMENTAL PROCEDURES was present in 8 ␮M concentration, and PEX19 was injected Protein Expression—Two human PEX3 fragments, compris- stepwise in 92 ␮M concentration. For binding studies between ing residues 26–373 and 41–373, were expressed in Escherichia sPEX3 and different PEX19-derived peptides, an ITC200 sys- coli. The corresponding DNA regions including a preceding tem (Microcal) was used. The protein was present in 17 ␮M at LIB4RI on December 18, 2018 tobacco etch virus protease cleavage site were cloned into the concentration, while the peptide fragments were injected step- ␮ vector pET32a (Novagen), which includes an N-terminal His6 wise (1 l/s) in a 5–15-fold higher concentration. ITC experi- tag. Site-directed mutagenesis (QuikChange௡, Stratagene) was ments with PEX19-derived peptides were performed at 25 °C in used to generate Cys-Ser mutations at position 235 in both buffer D (10 mM Na2HPO4, 1.8 mM KH2PO4, 140 mM NaCl, 2.7 cases.
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